Technical Field
The present invention relates to the technical field of pharmaceutical preparations, and particularly to a composition comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof, a preparation method therefor, a solid solution composition comprising high-purity (6S)-5-methyl tetrahydrofolic acid or a salt thereof, a preparation method therefor, and use of (6S)-5-methyl tetrahydrofolic acid and salts thereof, including the use of the composition or the solid solution comprising (6S)-5-methyl tetrahydrofolic acid or a salt thereof, in which a pharmaceutically acceptable reductive substance is added as a stabilizing agent. The safe method of using provides excellent safety properties of (6S)-5-methyl tetrahydrodrofolic acid products.
Related Art
In recent years, more and more research is conducted on the physiological activity for human of 5-methyl tetrahydrofolic acid and salts thereof. Good application prospects are exhibited when 5-methyl tetrahydrofolic acid or a salt thereof is used as a folic acid supplement to human for preventing and/or treating neurological disorders; used in combination with 5-fluorouracil or methotrexate for tumor therapy; used in the treatment of pathophysiology of vascular and cardiovascular diseases; and used in the therapy and adjuvant therapy of severe depression and severe senile dementia.
However, 5-methyl tetrahydrofolic acid or a salt thereof is quite labile, very susceptible to degradation, and highly sensitive particularly to oxygen and moisture. Therefore, a composition comprising 5-methyltetrahydrofolate or a salt thereof, such as a pharmaceutical preparation, generally suffers from the problems of poor stability, easy discoloration, darkened color, remarkably lowered content, and increased degradation product.
Many papers concern the oxidative degradation of 5-methyl tetrahydrofolic acid or its single active isomer (6S)-5-methyl tetrahydrofolic acid. Unfortunately, according to a study by the present inventors, many previous discussions on the primary oxidation products of 5-methyl tetrahydrofolic acid are either unilateral or even erroneous. These conclusions mislead people to reduce the quality requirements for 5-methyl tetrahydrofolic acid. Based on the discussions in Ratanasthien K et al. Serum folates in man (Ratanasthien K., Blair J. A., Leeming R. J., Cooke W. T. and Melikian V. (1977). J. Clin. Path. 30: 438-448); Blair J. A et al. Autoxidation of 5-Methyl-5,6,7,8-tetrahydrofolic acid (Blair J. A., Pearson A. J. and Robb A. J. (1975). J. C. S. Perkin II, p. 18-21.); and European Food Safety Authority, Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission related to Calcium L-Methylfolate Question (The EFSA Journal (2004) 135. 1-20), 5-methyl tetrahydrofolic acid is initially oxidized into 5-methyl dihydrofolic acid in the presence of an oxidant.

In other literatures, the primary oxidation product is characterized as 4-hydroxy-5-methyl tetrahydrofolic acid. This product is inferred to be not toxic according to the structure.
An efficient method is developed by the present inventors, in which a high-purity primary oxidation product of 5-methyl tetrahydrofolic acid is prepared firstly. A series of studies are conducted (PCT/CN2013/073959) and it is found unexpectedly that the primary oxidation product of 5-methyl tetrahydrofolic acid or a salt thereof is JK12A having a structural formula of

JK12A is a primary oxidation product of 5-methyl tetrahydrofolic acid, which, depending on specific conditions, has a relative retention time from the main peak of about 0.36 under the detection conditions of the present invention, and is also known as 5-methyl-dihydrofolic acid or 4-hydroxy-5-methyl-tetrahydrofolic acid in relevant literatures.
It is a product of new structure, and this finding overturns the previous conclusions, including those in the above-mentioned literatures. In general, large changes in structure will lead to a significant change in physiological activity. Subsequent studies show that JK12A has a significant inhibitory effect on T lymphocyte proliferation. It is generally believed that this causes the reduction of human immunity.
T cellTproliferationlymphocyteComprehensiveCellStimulusevaluationConcentrationMeanCellcontrolwith Con APercentdeterminedODSDsurvivalStimulusMean CPMSDenhancement/ControlM0.1220.003ratecontrol439778000inhibitionJK12A100.0000.0890.00273%40.0000.1090.00689%435374110−1%16.00.1130.00792%36061241−18%6.40.1030.00784%307843903−30%2.560.0760.00862%2550710004−42%1.0240.0730.01459%175908005−60%0.410.0520.01575%1407310299−68%0.164404597501−8%
An acute toxicity study of JK12A shows that at a dose of 2000 mg/kg, the test mice are killed on the spot within 1 minute. However, the anatomical findings show no significant changes in both the liver and the kidney, suggesting that the compound may possibly toxically target other tissues or organs and has a significant acute toxicity.
The present inventors find through studies that 5-methyl tetrahydropterioic acid is another common impurity in the product 5-methy tetrahydrofolic acid. Similar to the formation of JK12A, the impurity is oxidized to form JK1303 having a structure below

Subsequent studies have found that JK1303 has serious renal toxicity. 14 days after the mice are given a single dose of JK1303 at a dosage of 1000 mg/kg, severe renal tubular necrosis of the mice occurs, and a large number of vacuoles are anatomically found in the renal tubules. The toxicity of the primary oxidation product of 5-methyl tetrahydropterioic acid is also significantly changed.
The above experiments show that in a product containing 5-methyl tetrahydrofolic acid and its salt, the impurity JK12A produced from the oxidative degradation of 5-methyl tetrahydrofolic acid and its salt, and the oxidation product JK1303 of 5-methyl tetrahydropterioic acid are very harmful to human body, and this cannot and should not be ignored. Therefore, it is necessary to develop a solution for using 5-methyl tetrahydrofolic acid or its salt safely and reliably.
Chinese Patent No. CN102612358 discloses particles of stabilized salt of 5-methyl tetrahydrofolic acid, in which a crystalline salt of 5-methyl tetrahydrofolic acid is embedded in a wax to increase the stability.
Chinese Patent No. CN102813656 discloses a composition comprising stabilized salt of 5-methyl tetrahydrofolic acid, in which one or more amino acids free of mercapto groups are contained to increase the stability.
U.S. Pat. No. 6,441,168 discloses that the stability of 5-methyl tetrahydrofolic acid calcium salt is considerably increased by preparing it into a crystal form. However, the crystal form has a large particle size, is dissolved slowly, and needs to be ground first during the production of preparations, which trends to increase the risk of oxidation of 5-methyl tetrahydrofolic acid.
Chinese Patent No. CN2012086794 discloses a new crystal form of (6S)-5-methyl tetrahydrofolic acid calcium salt, which has good chemical stability and greatly increased solubility, such that no grinding is needed during the production of preparations. However, the content of the impurities such as JK12A is still high, and trends to increase gradually with the elapse of time.
It is also reported in literatures that 5-methyl tetrahydrofolic acid is embedded in microcapsules prepared with cellulose, shellac and other materials, to improve the stability of the active ingredients 5-methyl tetrahydrofolic acid in the preparation product.
However, the content of JK12A still cannot be controlled at a level that is safe enough by all these measures. For the raw medicine products of 5-methyl tetrahydrofolic acid commercially available at present, the total content of the impurities in a crystal form of 5-methyl tetrahydrofolic acid is generally in the range of 1-4%, in which the content of JK12A is up to 0.3-3.0%. In commercially available amorphous products, the total content of the impurities is as high as 10% or above, causing a serious threat to the safety of drugs during use. These facts show that the impurities including JK12A contained in all of these raw medicine products are not properly treated at present. The high chemical lability also makes it difficult to precisely control the dosage during use. For example, at a common dose of 0.451 mg, lability brings about a large deviation to the actual dose.